Diversity and Advantages of Culturable Endophytic Fungi from Tea (Camellia sinensis).

Sordariomycetes, Dothideomycetes, and Eurotioycetes are three classes of endophytes that colocalize with tea (Camellia sinensis). Overall, the diversity indexes in this study indicated a greater abundance of fungal endophytes in roots and stems. Taking the production system into account, conventional tea plantations exhibit lower diversity compared to organic tea plantations. Notably, the influence of agrochemicals had the largest impact on the fungal endophyte communities within roots and young leaves. Despite the limited fungal diversity in conventional plantations, three fungal endophytes were isolated from tea in this culture system: Diaporthe sp., YI-005; Diaporthe sp., SI-007; and Eurotium sp., RI-008. These isolated endophytes exhibited high antagonistic activity (93.00-97.00% inhibition of hypha growth) against Stagonosporopsis cucurbitacearum, the causal agent of gummy stem blight disease. On the other hand, endophytic fungi isolated from tea in an organic system-Pleosporales sp., SO-006 and Pleosporales sp., RO-013-established the ability to produce indole-3-acetic acid (IAA; 0.65 ± 0.06 µg/mL) and assist the solubilizing phosphorus (5.17 ± 1.03 µg/mL) from the soil, respectively. This suggested that the level of diversity, whether at the tissue level or within the farming system, did not directly correlate with the discovery of beneficial fungi. More importantly, these beneficial fungi showed the potential to develop into biological agents to control the devastating diseases in the cucurbit family and the potential for use as biofertilizers with a wide range of applications in plants. Therefore, it can be concluded that there are no restrictions limiting the use of fungal endophytes solely to the plant host from which they were originally isolated.

Xylaria feejeensis, SRNE2BP a Fungal Endophyte with Biocontrol Properties to Control Early Blight and Fusarium Wilt Disease in Tomato and Plant Growth Promotion Activity.

Over the past decade endophytic fungi have been known as a source of secondary metabolites with the ability to act as a biocontrol agents. Xylaria feejeensis, SRNE2BP a fungal endophyte isolated from a mangrove tree exhibited antagonistic activity against two fungal pathogens of tomato. Crude extract of X. feejeensis SRNE2BP significantly inhibited Fusarium oxysporum MFLUCC 19-0157 growth as shown approximately 60-75% in in vitro and in situ assays. Both assays showed that the endophyte also inhibited mycelium formation of Alternaria solani MFLUCC 19-0093 by 56% and 87%, respectively. The half maximal inhibitory concentration of X. feejeensis SRNE2BP crude extract against A. solani and F. oxysporum was approximately 7 mg/l. Crude extract and mycelium of X. feejeensis SRNE2BP showed potential in controlling early blight and fusarium wilt disease in tomato, respectively. Seedlings from seeds coated with crude extract of X. feejeensis SRNE2BP had lower disease severity (31.71%) of early blight disease compared to un-treated seeds (57.13%). Soil treated with 10% endophytic mycelium not only reduced fusarium wilt in tomato plant (55.55% severity compared with 91.66% in un-treated soil) but also promoted seed emergence and growth of tomato. Structure analysis revealed that 12 secondary metabolites, especially mellein derivatives, are major components of the crude extract of X. feejeensis SRNE2BP. These compounds could be responsible for antifungal activities; however, further study is required. Our findings strongly suggest that colonization with this fungal endophyte can be beneficial to the host plant especially with regards to plant growth promotion and broad antagonistic activity.

Discovery of the REN11 Locus From Vitis aestivalis for Stable Resistance to Grapevine Powdery Mildew in a Family Segregating for Several Unstable and Tissue-Specific Quantitative Resistance Loci.

Race-specific resistance loci, whether having qualitative or quantitative effects, present plant-breeding challenges for phenotypic selection and deciding which loci to select or stack with other resistance loci for improved durability. Previously, resistance to grapevine powdery mildew (GPM, caused by Erysiphe necator) was predicted to be conferred by at least three race-specific loci in the mapping family B37-28 × C56-11 segregating for GPM resistance from Vitis aestivalis. In this study, 9 years of vineyard GPM disease severity ratings plus a greenhouse and laboratory assays were genetically mapped, using a rhAmpSeq core genome marker platform with 2,000 local haplotype markers. A new qualitative resistance locus, named REN11, on the chromosome (Chr) 15 was found to be effective in nearly all (11 of 12) vineyard environments on leaves, rachis, berries, and most of the time (7 of 12) stems. REN11 was independently validated in a pseudo-testcross with the grandparent source of resistance, "Tamiami." Five other loci significantly predicted GPM severity on leaves in only one or two environments, which could indicate race-specific resistance or their roles in different timepoints in epidemic progress. Loci on Chr 8 and 9 reproducibly predicted disease severity on stems but not on other tissues and had additive effects with REN11 on the stems. The rhAmpSeq local haplotype sequences published in this study for REN11 and Chr 8 and 9 stem quantitative trait locus (QTL) can be used directly for marker-assisted selection or converted to SNP assays. In screening for REN11 in a diversity panel of 20,651 vines representing the diversity of Vitis, this rhAmpSeq haplotype had a false positive rate of 0.034% or less. The effects of the other foliar resistance loci detected in this study seem too unstable for genetic improvement regardless of quantitative effect size, whether due to race specificity or other environmental variables.

Napthoquinones from Neocosmospora sp.-Antibiotic Activity against Acidovorax citrulli, the Causative Agent of Bacterial Fruit Blotch in Watermelon and Melon.

Bacterial fruit blotch (BFB) is a bacterial disease that devastates Cucurbitaceae crops worldwide, causing significant economic losses. Currently, there is no means to treat or control the disease. This study focused on exploring the antibacterial properties of endophytic fungi against Acidovorax citrulli (Aac), the causative agent of BFB. Based on disc diffusion, time kill and MIC microdilution broth assays, four endophytes showed promise in controlling Aac. Nonetheless, only one strain, Neocosmospora sp. MFLUCC 17-0253, reduced the severity of disease on watermelon and melon seedlings up to 80%. Structure analysis revealed production of several compounds by the fungus. Three of these secondary metabolites, including mixture of 2-methoxy-6-methyl-7-acetonyl-8-hydroxy-1,4-maphthalenedione and 5,8-dihydroxy-7-acetonyl-1,4-naphthalenedione, anhydrojavanicin, and fusarnaphthoquinones B exhibited antagonistic activity against Aac. The chemical profile data in planta experiment analyzed by LC-Q/TOF-MS suggested successful colonization of endophytic fungi in their host plant and different metabolic profiles between treated and untreated seedling. Biofilm assay also demonstrated that secondary metabolites of Neocosmospora sp. MFLUCC 17-0253 significantly inhibited biofilm development of Aac. To the best of our knowledge, secondary metabolites that provide significant growth inhibition of Aac are reported for the first time. Thus, Neocosmospora sp. MFLUCC 17-0253 possesses high potential as a biocontrol agent for BFB disease.

A LysM Domain-Containing Protein LtLysM1 Is Important for Vegetative Growth and Pathogenesis in Woody Plant Pathogen Lasiodiplodia theobromae.

Lysin motif (LysM) proteins are reported to be necessary for the virulence and immune response suppression in many herbaceous plant pathogens, while far less is documented in woody plant pathogens. In this study, we preliminarily characterized the molecular function of a LysM protein LtLysM1 in woody plant pathogen Lasiodiplodia theobromae. Transcriptional profiles revealed that LtLysM1 is highly expressed at infectious stages, especially at 36 and 48 hours post inoculation. Amino acid sequence analyses revealed that LtLysM1 was a putative glycoprotein with 10 predicted N-glycosylation sites and one LysM domain. Pathogenicity tests showed that overexpressed transformants of LtLysM1 displayed increased virulence on grapevine shoots in comparison with that of wild type CSS-01s, and RNAi transformants of LtLysM1 exhibited significantly decreased lesion length when compared with that of wild type CSS-01s. Moreover, LtLysM1 was confirmed to be a secreted protein by a yeast signal peptide trap assay. Transient expression in Nicotiana benthamiana together with protein immunoblotting confirmed that LtLysM1 was an N-glycosylated protein. In contrast to previously reported LysM protein Slp1 and OsCEBiP, LtLysM1 molecule did not interact with itself based on yeast two hybrid and co-immunoprecipitation assays. These results indicate that LtLysM1 is a secreted protein and functions as a critical virulence factor during the disease symptom development in woody plants.

Antifungal activity of 8-methoxynaphthalen-1-ol isolated from the endophytic fungus Diatrype palmicola MFLUCC 17-0313 against the plant pathogenic fungus Athelia rolfsii on tomatoes.

Thirty-four endophytic fungal isolates were obtained from the leaves of the medicinal plant Polyscias fruticosa, and their antagonistic activities against the growth of the common tomatoes plant pathogenic fungus Athelia rolfsii were initially screened using a dual culture assay. The endophytic fungus MFLUCC 17-0313, which was later molecularly identified as Diatrype palmicola, displayed the highest inhibition percentage (49.98%) in comparison to the others. This fungus was then chosen for further evaluation. Its culture broth and mycelia from a 10 L scale were separated and extracted using ethyl acetate, methanol, and hexane. Each extract was tested for antifungal activity against the same pathogen using a disc diffusion assay. Only the crude hexane extract of fungal mycelium showed antifungal activity. The hexane extract was fractioned using sephadex gel filtration chromatography and each fraction was tested for antifungal activity until the one with the highest inhibition percentage was obtained. The bioactive compound was identified as 8-methoxynaphthalen-1-ol using nuclear magnetic resonance spectroscopy and mass spectrometry. The minimum inhibition concentration of 8-methoxynaphthalen-1-ol was demonstrated at 250 µg/mL against the selected pathogen. Using the leaf assay, the solution of 8-methoxynapthalen-1-ol was tested for phytotoxic activity against A. rolfsii and was found to have no phytotoxic effects. These results showed that 8-methoxynaphthalen-1-ol has the potential for controlling the growth of A. rolfsii, the cause of Southern blight disease on tomatoes. This study may provide the foundation for future use of this compound as a biofungicide.

High Genetic Diversity and Species Complexity of Diaporthe Associated With Grapevine Dieback in China.

Grapevine trunk diseases have become one of the main threats to grape production worldwide, with Diaporthe species as an emerging group of pathogens in China. At present, relatively little is known about the taxonomy and genetic diversity of Chinese Diaporthe populations, including their relationships to other populations worldwide. Here, we conducted an extensive field survey in six provinces in China to identify and characterize Diaporthe species in grape vineyards. Ninety-four isolates were identified and analyzed using multi-locus phylogeny. The isolates belonged to eight species, including three novel taxa, Diaporthe guangxiensis (D. guangxiensis), Diaporthe hubeiensis (D. hubeiensis), Diaporthe viniferae (D. viniferae), and three new host records, Diaporthe gulyae (D. gulyae), Diaporthe pescicola (D. pescicola), and Diaporthe unshiuensis (D. unshiuensis). The most commonly isolated species was Diaporthe eres (D. eres). In addition, high genetic diversity was observed for D. eres in Chinese vineyards. Haplotype network analysis of D. eres isolates from China and Europe showed a close relationship between samples from the two geographical locations and evidence for recombination. In comparative pathogenicity testing, D. gulyae was the most aggressive taxon, whereas D. hubeiensis was the least aggressive. This study provides new insights into the Diaporthe species associated with grapevines in China, and our results can be used to develop effective disease management strategies.

The OmpA Gene of Xanthomonas axonopodis pv. glycines is Involved in Pathogenesis of Pustule Disease on Soybean.

The goal of this study was to elucidate the role of the outer membrane protein A (ompA) gene of Xanthomonas axonopodis pv. glycines in bacterial pustule pathogenesis of soybean. An ompA mutant of X. axonopodis pv. glycines KU-P-SW005 was shown to significantly decrease cellulase, pectate lyase, and polysaccharide production. The production of these proteins in the ompA mutant was approximately five times lower than that of the wildtype. The ompA mutant also exhibited modified biofilm development. More importantly, the mutant reduced disease severity to the soybean. Ten days after inoculation, the virulence rating of the susceptible soybean cv. SJ4 inoculated with the ompA mutant was 11.23%, compared with 87.98% for the complemented ompA mutant. Production of cellulase, pectate lyase, polysaccharide was restored, biofilm, and pustule numbers were restored in the complemented ompA mutant that did not differ from the wild type. Taken together, these data suggest that OmpA-mediated invasion plays an important role in protein secretion during pathogenesis to soybean.

Two new entomopathogenic species of Ophiocordyceps in Thailand.

Ophiocordyceps is entomopathogenic and the largest studied genus in the family Ophiocordycipitaceae. Many species in this genus have been reported from Thailand. The first new species introduced in this paper, Ophiocordycepsglobiceps, differs from other species based on its smaller perithecia, shorter asci and secondary ascospores and additionally, in parasitising fly species. Phylogenetic analyses of combined LSU, SSU, ITS, TEF1α and RPB1 sequence data indicate that O.globiceps forms a distinct lineage within the genus Ophiocordyceps as a new species. The second new species, Ophiocordycepssporangifera, is distinguished from closely related species by infecting larvae of insects (Coleoptera, Elateridae) and by producing white to brown sporangia, longer secondary synnemata and shorter primary and secondary phialides. We introduce O.sporangifera based on its significant morphological differences from other similar species, even though phylogenetic distinction is not well-supported.

Evaluation of key meteorological determinants of wintering and flowering patterns of five rubber clones in Xishuangbanna, Yunnan, China.

All rubber tree clones (Hevea brasiliensis) exhibit regular annual wintering characterized by senescence and abscission of leaves. After 3-4 weeks, this is followed by the onset of new leaves. It is likely that the timing of leaf onset affects the susceptibility of rubber trees to rubber powdery mildew disease, as this predominantly infests young leaves. However, little information is available on the phenological behavior of different rubber clones, or how meteorological factors affect such behavior. We assessed the wintering and flowering patterns of five rubber clones in Xishuangbanna, southwest China, based on observations made from 1978 to 2011, and evaluated how these patterns responded to different meteorological factors. Partial least squares regression was used to analyze the timing of defoliation, refoliation, and flowering. Our results showed that the two clones RRIM 600 and GT1 defoliated during the last week of December and refoliated in the last week of January, and clones Yunyan 277-5, Yunyan 34-4, and PR 107 defoliated during the first week of January and refoliated in the second week of February. The number of hours of sunshine during both the rainy season and the cold dry period in the dry season were important determinants of phenological changes in the rubber trees. Similarly, higher temperatures tended to delay the onset of defoliation and refoliation, and were a triggering factor for the onset of flowering. These results may help rubber cultivators to schedule appropriate disease control measures, as well as to design hybridization programs aiming at the production of clones which are resistant to foliar disease.

Multigene phylogenetics of Polycephalomyces (Ophiocordycipitaceae, Hypocreales), with two new species from Thailand.

Polycephalomyces (Ophiocordycipitaceae) species are found in subtropical regions and are parasitic or hyperparasitic on insects. Two new species, P. aurantiacus and P. marginaliradians, parasitic on Ophiocordyceps barnesii and larva of Cossidae respectively, are introduced in this paper. Morphological comparison with extant species and DNA based phylogenies from analyses of a multigene dataset support the establishment of the new taxa. Polycephalomyces aurantiacus, exhibiting a hyperparasitic lifestyle on Ophiocordyceps barnesii, differs from other species in producing orange conidia in mass and have longer ß-phialides in culture. Polycephalomyces marginaliradians differs from other Ophiocordyceps species by producing single stromata with a stipe, smaller perithecia and branched α-phialides and catenate α-conidia and is parasitic on Cossidae. A combined nrSSU, nrLSU, ITS, tef-1a, rpb1 and rpb2 sequence data was analysed phylogenetically including Ophiocordyceps and Polycephalomyces taxa. The new species described herein are clearly distinct from other species in Polycephalomyces. We provide a key to the species of Polycephalomyces and discuss relevant interspecies relationships.

Morpho-Molecular Characterization of Two Ampelomyces spp. (Pleosporales) Strains Mycoparasites of Powdery Mildew of Hevea brasiliensis.

Powdery mildew disease of rubber affects immature green leaves, buds, inflorescences, and other immature tissues of rubber trees, resulting in up to 45% losses in rubber latex yield worldwide. The disease is often controlled by dusting the diseased plants with powdered sulfur, which can have long-term negative effects on the environment. Therefore, it is necessary to search for alternative and environmentally friendly control methods for this disease. This study aimed to identify mycoparasites associated with rubber powdery mildew species, and characterize them on the basis of morpho-molecular characteristics and phylogenetic analyses of ITS rDNA regions. We observed that the Ampelomyces fungus parasitizes rubber powdery mildew, and eventually destroys it. Furthermore, on the basis of phylogenetic analyses and morphological characteristics we confirmed that the Ampelomyces mycoparasite isolated from rubber powdery mildew is closely related to other mycohost taxa in the Erysiphe genus. A total of 73 (71 retrieved from GenBank and two obtained from fresh collections of rubber powdery mildew fungi) Ampelomyces spp. were analyzed using ITS rDNA sequences and 153 polymorphic sites were identified through haplotypic analyses. A total of 28 haplotypes (H1-H28) were identified to have a complex network of mutation events. The results from phylogenetic tree constructed on the basis of maximum likelihood analyses, and the haplotype network tree revealed similar relationships of clustering pattern. This work presents the first report on morpho-molecular characterization of Ampelomyces species that are mycoparasites of powdery mildew of Hevea brasiliensis.

Multiple gene genealogy reveals high genetic diversity and evidence for multiple origins of Chinese Plasmopara viticola population.

Downy mildew caused by Plasmopara viticola is one of the most devastating diseases of grapevines worldwide. So far, the genetic diversity and origin of the Chinese P. viticola population are unclear. In the present study, 103 P. viticola isolates were sequenced at four gene regions: internal transcribed spacer one (ITS), large subunit of ribosomal RNA (LSU), actin gene (ACT) and beta-tubulin (TUB). The sequences were analyzed to obtain polymorphism and diversity information of the Chinese population as well as to infer the relationships between Chinese and American isolates. High genetic diversity was observed for the Chinese population, with evidence of sub-structuring based on climate. Phylogenetic analysis and haplotype networks showed evidence of close relationships between some American and Chinese isolates, consistent with recent introduction from America to China via planting materials. However, there is also evidence for endemic Chinese P. viticola isolates. Our results suggest that the current Chinese Plasmopara viticola population is an admixture of endemic and introduced isolates.

Antibacterial and Antioxidant Constituents of Extracts of Endophytic Fungi Isolated from Ocimum basilicum var. thyrsiflora Leaves.

Fourteen fungal endophytes were isolated from the Ocimum basilicum var. thyrsiflora leaves collected from Northern Thailand. Eight genera were identified including Aspergillus, Ascochyta, Nigrospora, Blastomyces, Colletotrichum, Exidia, Clitopilus, and Nomuraea. The antibacterial activity of crude extracts from all endophytic fungi was tested against nine human bacterial pathogens: Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Bacillus cereus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Vibrio cholerae, and Vibrio parahaemolyticus. All crude extracts showed some degree of antibacterial activity, but the crude extract from Nigrospora MFLUCC16-0605 exhibited broad spectrum activity with MIC values ranging from 7.81 to 250 µg/mL. The antioxidant activity of all crude extracts was also investigated by DPPH radical scavenging assay. Crude extract from MFLUCC16-0605 had high antioxidant activity (IC50 value of 15.36 µg/mL) comparable to the trolox and gallic acid standards showing IC50 values of 2.56 and 12.89 µg/mL, respectively. The chemical composition of the crude extract from MFLUCC16-0605 was determined using GC-MS. Sixty-two compounds were identified representing 92.09% of crude extract with six major components including 5E,9E-farnesyl acetone, columellarin, totarene, laurenan-2-one, and 8S,13-cedranediol. PCR amplification and sequencing of the barcoding region identified MFLUCC16-0605 as belonging to Nigrospora genus. The notable activities of MFLUCC16-0605 indicate that the endophyte is a potent natural resource and its use as an antibacterial/antioxidant agent should be further explored.

Taxonomic revision and phylogenetic analyses of rubber powdery mildew fungi.

Powdery mildew is a fungal disease that infects a wide range of plants, including rubber trees, which results in a reduction of latex yields of up to 45%. The causal agent of powdery mildew of rubber was first described as Oidium heveae, but later morpho-molecular research suggested that in the past, O. heveae has been confused with Erysiphe quercicola. However, it is still under debate whether the causal agent should be classified as a species of the genus Erysiphe emend. or Golovinomyces and Podosphaera, respectively. Therefore, the aim of this study was to undertake the morpho-molecular characterization of powdery mildew species associated with rubber trees, thus resolving these taxonomic issues. Morphological observation under light and scanning electron microscopes (SEM) clearly identified two morphotypes of the rubber powdery mildew. With the support of morphological and phylogenetic data, one of the two morphotypes was identified as the asexual morph of E. quercicola, while the second morphotype is still insufficiently known and according to the morphological results obtained we assume that it might belong to the genus Golovinomyces. More collections and additional molecular data are required for final conclusions regarding the exact taxonomic position of the second morphotype of rubber powdery mildew and its relation to the name O. heveae. The haplotype analysis identified eight haplotype groups of E. quercicola indicating the high genetic diversity of the species.

Grapevine powdery mildew resistance and susceptibility loci identified on a high-resolution SNP map.

Improved efficacy and durability of powdery mildew resistance can be enhanced via knowledge of the genetics of resistance and susceptibility coupled with the development of high-resolution maps to facilitate the stacking of multiple resistance genes and other desirable traits. We studied the inheritance of powdery mildew (Erysiphe necator) resistance and susceptibility of wild Vitis rupestris B38 and cultivated V. vinifera 'Chardonnay', finding evidence for quantitative variation. Molecular markers were identified using genotyping-by-sequencing, resulting in 16,833 single nucleotide polymorphisms (SNPs) based on alignment to the V. vinifera 'PN40024' reference genome sequence. With an average density of 36 SNPs/Mbp and uniform coverage of the genome, this 17K set was used to identify 11 SNPs on chromosome 7 associated with a resistance locus from V. rupestris B38 and ten SNPs on chromosome 9 associated with a locus for susceptibility from 'Chardonnay' using single marker association and linkage disequilibrium analysis. Linkage maps for V. rupestris B38 (1,146 SNPs) and 'Chardonnay' (1,215 SNPs) were constructed and used to corroborate the 'Chardonnay' locus named Sen1 (Susceptibility to Erysiphe necator 1), providing the first insight into the genetics of susceptibility to powdery mildew from V. vinifera. The identification of markers associated with a susceptibility locus in a V. vinifera background can be used for negative selection among breeding progenies. This work improves our understanding of the nature of powdery mildew resistance in V. rupestris B38 and 'Chardonnay', while applying next-generation sequencing tools to advance grapevine genomics and breeding.